Various scientific articles and patents are referred to in parentheses throughout the specification. These documents are incorporated by reference herein to describe the state of the art to which this invention pertains. Full citations of the scientific articles appear at the end of the specification.
Turfgrasses and turfgrass breeding are of significant economic importance worldwide. In recent years, traditional breeding programs have been augmented by molecular biological and recombinant techniques. However, similar to most monocotyledenous plants, turfgrasses have proven recalcitrant to tissue culture, transformation and regeneration procedures. Among these, Agrobacterium-mediated transformation of a turfgrass heretofore has not been accomplished.
Turfgrass transformation has been achieved using direct methods of DNA transfer, including protoplast transformation and particle gun bombardment. Nonetheless, Agrobacterium-mediated transformation offers several advantages over particle gun bombardment or other means of direct gene transfer. These include stable transgene integration without rearrangement of either host or transgene DNA; preferential integration of the transgene into transcriptionally active regions of the genome; ability to transfer large pieces of DNA; and integration of low numbers of gene copies into plant nuclear DNA which is particularly important to minimize possible co-suppression of the transgene in later generations.
Until recently, Agrobacterium-mediated transformation was thought to be limited to dicotyledonous plants. However, Hiei et al. in 1994 described efficient transformation of rice by Agrobacterium, and subsequently there have been convincing reports for maize, barley and wheat (Ishida et al., 1996; Tingay et al., 1997; Cheng et al., 1997; see also U.S. Pat. No. 5,591,616 to Hiei et al). Numerous factors are of critical importance in Agrobacterium-mediated transformation of monocots, including the type and stage of tissue that is infected, the vector and bacterial strains used, plant genotype, tissue culture conditions, and the actual infection process. As a result, methods that have proven successful for Agrobacterium-mediated transformation of some monocots, such as rice and maize, have not been successful for transforming turfgrass.
An object of the present invention is to develop an efficient and reliable transformation system for turfgrass, mediated by Agrobacterium tumefaciens. Another object of the invention is to regenerate transgenic plants containing one or more foreign genes introduced by Agrobacterium tumefaciens-mediated transformation.